International Journal of Molecular Sciences Review The Human Tissue-Engineered Cornea (hTEC): Recent Progress Louis-Philippe Guérin 1,2,3, Gaëtan Le-Bel 1,2,3,4, Pascale Desjardins 1,2,3,4, Camille Couture 1,2,3,4, Elodie Gillard 1,2,3, Élodie Boisselier 1,2,3 , Richard Bazin 1,2,3, Lucie Germain 1,2,3,4 and Sylvain L. Guérin 1,2,3,* 1 CUO-Recherche, Médecine Régénératrice—Centre de Recherche du CHU de Québec, Université Laval, Québec, QC G1S 4L8, Canada; [email protected] (L.-P.G.); [email protected] (G.L.-B.); [email protected] (P.D.); [email protected] (C.C.); [email protected] (E.G.); [email protected] (É.B.); [email protected] (R.B.); [email protected] (L.G.) 2 Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC G1J 1Z4, Canada 3 Département d’Ophtalmologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada 4 Département de Chirurgie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada * Correspondence: [email protected]; Tel.: +1-418-682-7565 Abstract: Each day, about 2000 U.S. workers have a job-related eye injury requiring medical treatment. Corneal diseases are the fifth cause of blindness worldwide. Most of these diseases can be cured using one form or another of corneal transplantation, which is the most successful transplantation in humans. In 2012, it was estimated that 12.7 million people were waiting for a corneal transplantation worldwide. Unfortunately, only 1 in 70 patients received a corneal graft that same year. In order to provide alternatives to the shortage of graftable corneas, considerable progress has been achieved in the development of living corneal substitutes produced by tissue engineering and designed to mimic their in vivo counterpart in terms of cell phenotype and tissue architecture. Most of these substitutes use synthetic biomaterials combined with immortalized cells, which makes them dissimilar from the Citation: Guérin, L.-P.; Le-Bel, G.; native cornea. However, studies have emerged that describe the production of tridimensional (3D) Desjardins, P.; Couture, C.; Gillard, E.; tissue-engineered corneas using untransformed human corneal epithelial cells grown on a totally Boisselier, É.; Bazin, R.; Germain, L.; Guérin, S.L. The Human natural stroma synthesized by living corneal fibroblasts, that also show appropriate histology and Tissue-Engineered Cornea (hTEC): expression of both extracellular matrix (ECM) components and integrins. This review highlights Recent Progress. Int. J. Mol. Sci. 2021, contributions from laboratories working on the production of human tissue-engineered corneas 22, 1291. https://doi.org/10.3390/ (hTECs) as future substitutes for grafting purposes. It overviews alternative models to the grafting of ijms22031291 cadaveric corneas where cell organization is provided by the substrate, and then focuses on their 3D counterparts that are closer to the native human corneal architecture because of their tissue Academic Editor: Claudiu T. Supuran development and cell arrangement properties. These completely biological hTECs are therefore Received: 22 December 2020 very promising as models that may help understand many aspects of the molecular and cellular Accepted: 19 January 2021 mechanistic response of the cornea toward different types of diseases or wounds, as well as assist in Published: 28 January 2021 the development of novel drugs that might be promising for therapeutic purposes. Publisher’s Note: MDPI stays neutral Keywords: human cornea; tissue-engineering; 3D corneal model; wound healing; limbal stem cells; with regard to jurisdictional claims in epithelium; stroma; endothelium published maps and institutional affil- iations. 1. Introduction Vision is crucial when it comes to our interactions with our surroundings. In order Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. to see, light must be refracted by the cornea, the outer layer of the eye, onto the lens, and This article is an open access article then reach the retina. Thus, our visual abilities rely heavily on corneal transparency and distributed under the terms and its refractive power. The cornea accounts for two-thirds of the overall refractive power of conditions of the Creative Commons the eye and provides defense against trauma and infections. It is composed of three layers: Attribution (CC BY) license (https:// the epithelium, the stroma, and the endothelium, which are divided by two extracellular creativecommons.org/licenses/by/ matrix interfaces: the Bowman’s layer and the Descemet membrane [1]. The epithelium 4.0/). can readily regenerate and restore normal vision after a minor trauma. However, in the Int. J. Mol. Sci. 2021, 22, 1291. https://doi.org/10.3390/ijms22031291 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 45 matrix interfaces: the Bowman’s layer and the Descemet membrane [1]. The epithelium Int. J. Mol. Sci. 2021, 22, 1291 can readily regenerate and restore normal vision after a minor trauma. However,2 ofin 43 the case of deeper wounds, the stroma tends to develop permanent opaque scars, the extent of which is related to the severity of the injury. The endothelium cannot regenerate in humanscase of deeperunder normal wounds, conditions. the stroma tendsThe principal to develop physiological permanent opaque function scars, of these the extent cells ofis to allowwhich the is leakage related to of the solutes severity and of nutrients the injury. from The endotheliumthe aqueous cannothumorregenerate to the more in superficial humans layersunder of normal the cornea, conditions. while The at principalthe same physiological time pumping function water of in these the cells opposite is to allow direction. the Whenleakage endothelial of solutes cells and are nutrients damaged, from the the corn aqueousea swells, humor loses to the transparency, more superficial and develops layers bullaeof the on cornea, its anterior while surface. at the same time pumping water in the opposite direction. When endothelial cells are damaged, the cornea swells, loses transparency, and develops bullae 1.1.on Anatomy its anterior of the surface. Human Cornea 1.1.The Anatomy human of thecornea Human is a Cornea transparent avascular tissue and is the most anterior structure of the eye.The humanIts three cornea main ispurposes a transparent are to avascular protect the tissue intraocular and is the contents, most anterior to allow structure light to enterof the the eye. eye Its and three reach main the purposes retina, and are to to provid protecte thetwo intraocular thirds of the contents, refractive to allowpower light of the opticto enter system the eye[2]. andMany reach factors the retina,contribute and toto providecorneal twotransparency: thirds of the the refractive regularity power of the surfaceof the opticand thickness system [2 ].of Manythe epithelium factors contribute in association to corneal with transparency: the integrity the of regularitythe lacrimal film,ofthe the surfaceregular andarchitecture thickness of of the the collagen epithelium fibrils in in association the stroma, with the theproduction integrity of of soluble the crystallinlacrimal film,proteins the regularand the architecture presence of of theproteoglycans collagen fibrils produced in the stroma, by the the stromal production kerato- cytes,of soluble the regulation crystallin of proteins the hydration and the presencelevel by the of proteoglycans endothelium, produced and the absence by the stromal of vascu- larizationkeratocytes, [3–7]. the The regulation diameter of of the the hydration cornea is level approximately by the endothelium, 12 mm and and the the mean absence anterior of cornealvascularization curvature [ 3radius–7]. The is diameteraround 8 ofmm the [8]. cornea The iscorneal approximately thickness 12 is mmabout and 540 the µm mean in the centeranterior and corneal 700 µm curvature in the radius periphery. is around Being 8 mm avascular, [8]. The corneal the cornea’s thickness nutritional is about 540 supplyµm µ (mostlyin the centeroxygen and and 700 glucose)m in the co periphery.mes from small Being blood avascular, vessels the cornea’sin the corneoscleral nutritional supply junction (mostly oxygen and glucose) comes from small blood vessels in the corneoscleral junction as well as from palpebral arteries through the aqueous humor and the lacrimal film [1,9]. as well as from palpebral arteries through the aqueous humor and the lacrimal film [1,9]. Moreover, it is estimated that the cornea has an innervation density 300–600 times that of Moreover, it is estimated that the cornea has an innervation density 300–600 times that of thethe skin skin and and 20–40 20–40 times times that that of of the the tooth pulp,pulp, makingmaking it it one one of of the the most most innerved innerved tissues tissues inin the the human human body body [10,11]. [10,11]. The The five five components components ofof thethe cornea cornea (from (from anterior anterior to to posterior: posterior: cornealcorneal epithelium, epithelium, Bowman’s Bowman’s membrane, cornealcorneal stroma,stroma, Descemet’s Descemet’s membrane, membrane, and and cornealcorneal endothelium) endothelium) are are described described belowbelow [[2]2] (also(also refer refer to to Figure Figure1). 1). FigureFigure 1. Schematic 1. Schematic of the of thehuman human cornea cornea and and histology. histology. LeftLeft panel:panel: Schematic Schematic view view of the of thehuman human eye. eye. The Thecornea cornea forms theforms transparent the transparent front part front of the part eyeball. of the Central eyeball. panel:Central Frompanel: anterior From anteriorto posterior, to posterior, the cornea the is cornea made is up made of aup stratified of a squamousstratified epithelium squamous epitheliumdeposited depositedon a basement on a basement membrane, membrane, follows follows the Bowman’s the Bowman’s membrane, membrane, a stroma, a stroma, composed composed pre- predominantly of collagen fibrils in which keratocytes are entangled, the Descemet’s membrane, and a monolayer of endothelial cells.